BBli 



UNITED STATES DEPARTMENT OF AGRICULTURE 
BULLETIN No. 289 

Contribntion from the Bnreaa of Plant Indnstr; 
WM. A. TAYLOR, Chief 




Washington, D. C. 



PROFESSIONAL PAPER 



September 21, 1915 



RED-CLOVER SEED PRODUCTION: 

POLLINATION STUDIES 

By 

J. M. WESTGATE, Agronomist, and H. S. COE, Scientific Assistant, Office 

of Forage-Crop Investigations, in Collaboration with A. T. WIANCKO 

and F. E. ROBBINS, of the Indiana Agricultural Experiment 

Station, and H. D. HUGHES, L. H. PAMMEL, and 

J. N. MARTIN, of the Iowa Agricultural 

Experiment Station 



CONTENTS 



Introduction . . . . ~. ' 

Previous Investigations on the Pollina- 
tion of Red Clover 

Outline of Pollinating Experiments . . 

Structure of the Red-Clover Flower . . 

Length of the Corolla Tube of Red-Clover 
Flowers . . . 

Development of the Flowers of Red 
Clover 

Fertilization of Red-Clover Flowers . . 

Potency of Pollen in Self-pollination . . 



Page 
1 

2 
5 



Page 



Cross-Pollination and Self-Pollination of 

Red Clover 11 

Artificial Manipulation of Clover Heads . 12 
Bumblebees as Cvoss-PoUinaters of Red 

Clover 17 

Honeybees as Crfcss-Pollinators of Red 

Clover 18 

Mechanical Cross-PoIUnators of Red 

Clover 20 

Summary 26 

Literature Cited 29 




WASHINGTON 

GOVERNMENT PRINTING OFFICE 

1915 



3:P 24 iSIS 



UNITED STATES DEPARTMENT OF AGRICULTURE 

BULLETIN No. 289 

Contribution from the Bureau of Plant Industry 
WM. A. TAYLOR, Chief 





Washington, D. C. 



PROFESSIONAL PAPER 



September 21, 1915 



RED-CLOVER SEED PRODUCTION: POLLINATION 

STUDIES. 

By J. M. ^yESTGATE, Agronomist, and H. S. Voe, Scientijic Assistant, Ojjice of Forage- 
Crop Investigations. 

In collaboration with A. T. Wiancko and F. E. Robbins, of the Indiana Agricullnral 
Experiment Station, and H. D. Hughes, L. H. Pammel, and J. N. Martin, of the 
Iowa Agricultural Experiment Station. 



CONTENTS. 



Introduction 1 

Previous investigations on the pollination of 

red clover 2 

Outline of pollinating experiiTients 5 

Structtire of the red-clover flower 5 

Length of the corolla tube of red-clover flowers. 7 

Development of the flowers of red clover 7 

Fertilization of red-clover flowers 10 

Potency of pollen in self-pollination 10 



Page. 
Cross-pollination and self-pollination of red 

clover 11 

Artificial manipulation of clover heads 12 

Bumblebees as cross-pollinators of redclover 17 

Honeybees as cross-pollinators of red clover 18 

Mechanical cross-polluiators of red clover 20 

Summary 2G 

Literature cited 29 



INTRODUCTION. 

For a number of years the quantity of seed of red clover (Trifolium 
pmtcnse) produced in this country has been insufficient to supply the 
demand for reseeding purposes in the clover-belt States. This not 
only has caused the seed to be high in price, but has resulted in the 
importation of large quantities of foreign seed, some of whicli, on 
account of the impurities present and its low vitality, has been 
considerably loss desirable than the ordinary home-grown strains. ^ 

The prime importance of clover in the ordinary farm rotations m 
the corn and clover belt States makes the continued maintenance of 
the clover acreage of great moment to the agricultural prosperity of 
the country. This problem has been approached from four different 
angles. First, to determine the minimum amount of seed necessary 
to obtain a stand, so that much less than the quantity of seed ordi- 
narily sown will be sufficient to produce a satisfactory yield, for any 

2990°— 3uU, 2S9— 15 1 



2 BULLETIN 289, U. S. DEPAETMENT OF AGEICULTUEE. 

reduction in tlie quantity of seed required to sow an acre will propor- 
tionately increase the acre'age throughout the country which can be 
so■v^^l with the available supply of seed. The second line of attack 
has been to determine the environmental conditions necessary to 
the maintenance of a satisfactory stand of clover. That these con- 
ditions are less favorable than they have been in the past is 
indicated by the increasing difficulty experienced by many farmers 
in mamtaining clover in the ordinary rotations. A third line of 
attack has been the possibility of developing a heavy-seeding, 
hardy strain of clover with good forage and hay producing qualities. 
The fourth line of attack, the one with which the present publication is 
concerned, has been a study of various means of affecting the yield of 
clover seed under field conditions as they exist tliroughout the 
clover-growing sections of the country. One phase of this work has 
been to determine the effect of the time of cutting or clipping the 
first growth on the seed production of the subsequent crop. The 
second phase, that with which this bulletin is primarily concerned, 
has been the effect of various mechanical forms of pollination upon 
the quantity of seed produced. / , 

PREVIOUS INVESTIGATIONS ON THE POLLINATION OF RED CLOVER. 

Since the time of the publication of the statement by Darwin (6, 
p. 361; 7, p. 90)' that 100 heads of red clover on plants protected 
from insects during the blooming period did not produce a single seed 
while a similar number of heads exposed to insects produced an 
average of 27 seeds per head, many scientists have investigated this 
subject. Knuth (22, v. 1, p. 36-37; v. 2, p. 289) accepts Dai-win's 
experimental results and states that red clover, crimson clover, and 
white clover are among the best examples of self-sterility in plants. 
Stebler and Schroter (39, p. 123) in discussing the polhnation of red 
clover say that there is no experimental evidence to show that pollen 
from a flower can not, when applied to its own stigma, fertihze the 
ovules, but they also state that pollen wliicli is effective in producing 
fertihzation has in all probabihty come from some other flower. The 
same authors (40, p. 14, 122) in a later edition state that red clover 
is self -sterile. Frandsen, according to Lindhard (23), found red 
clover to be practically self-sterile. From 1,235 flowers in 1910 and 
1,305 flowers in 1911, wliich were self-polhnated by Frandsen, 0.07 
per cent set seed. In 1910 Frandsen pollinated 1,488 flowers and in 
1911, 1,455 flowers with pollen from other heads on the same plants; 
0.8 per cent of the flowers set seed in 1910 and 0.4 per cent in 1911. 

Wallace (43, p. 121) states that insects must perform the indispen- 
sable work of cross-polHnating red clover, but later says (44) that he 
has been incHned to think that climatic conditions rather than the 



> Reference is made by number to " Literatu'e cited," p. 29. 



RED-CLOVER SEED PRODUCTION. 6 

presence or absence of insects influence seed production. The work 
of Sirrine (37, p. 89-90) , as well as that of Wltte (46) , showed red clover 
to be self -sterile. In the experiments of Cook (5), Shamel (36), and 
Kirchner (21) no seed was produced when heads were covered before 
blooming and not pollinated. Fruwirth (11; 12, p. 163-166) did not 
obtain a single seed when heads under cover were left undisturbed 
or when they were pollinated with pollen from another head on the 
same plant, while heads polUnated with pollen from another plant 
produced seed. Bolley (4) obtained but two seeds from one head 
of a large area which was placed under a fine screen before any of the 
flowers came into bloom. He states that insects other than bumble- 
bees must pollinate the flowers, since the bumblebees were scarce 
and the clover set well. Genevier (13) states that the fertilization 
of clover does not depend on the presence of bumblebees. Pammel 
and King (32) report but two seeds from 643 heads which were 
allowed to mature under a screen cover, while Washburn (45) says 
that only by the aid of bumblebees was he able to obtain seed. 

Armstrong (L), in writing about New Zealand, says there is every 
reason to beheve that numerous individuals belonging to Trifolium 
pratense are self-fertile and that they produce seK-fertile progeny. 
According to him the American strain is usually, if not always, self- 
fertile. McAlpine (24) discusses Garton's experiments, which show 
that the self-fertihzing property is as common with red clover as it is 
with the bean. The following is quoted from Kerner (20, p. 407): 
"Pisum and Ervum, Lotus and Melilotus, the various species of Tri- 
folium, almost all of them, when un visited by insects, ripen seed, 
only a few species here and there bemg infertile when dependent 
upon their own resources." Nothing definite can be taken from 
Kerner's statement, since he does not quote any species or give defi- 
nite exceptions to his statement. Hopkins (14) says he is not ready 
to admit that self-fertilization does not take place and that he is 
inclined to believe a crop of seed can be grown without the aid of 
bumblebees. The same author (15, p. 73) states that honeybees 
serve the same purpose as bumblebees in cross-fertilizing red clover. 
The work of Beal (2, p. 325-328) shows that bumblebees increased 
the seed production about four times, since in a check cage he received 
25 seeds from 50 heads, while in the cage where bumblebees were 
placed 94 seeds from 50 heads were obtained. Martinet (27) found 
red clover to be self-fertile, stating that cross-pollination might 
have been brought about by very small insects (undoubtedly mean- 
ing thrips). Fruwirth (12, p. 163-166), however, showed that 
thrips transferred from other clover fields in large numbers produced 
no seed in his experiments. Meehan (28) states that a careful exami- 
nation of the clover flower in all its stages convinced him that from 
its structure and behavior it was self-fertile. It is still an open 



4 BULLETIN 289, U. S. DEPARTMENT OF AGRICULTURE. 

question whether or not red clover is self -fertile, according to Smith 
(38, p. 236). 

Garton, according to Wallace (44), claims that red-clover flowers 
are cleistogamous, but Martin (26) in his work on the cytology of 
red-clover flowers disproved this theory. Garton attempts to prove 
that the flowers are cleistogamous by saying that the ovules are 
well formed by the time the flowers open. The ovary is quite large 
at this time, and it was undoubtedly taken to be a developing ovule. 
Pammel and King (32) record that self-fertilization was accomplished 
in some experiments at Ames, Iowa, by irritating the stigmas. Hunt 
(19) speaks as follows: "It has long been recognized that red-clover 
and other leguminous flowers may be self -pollinated, although it 
has never been determined whether self-pollination or cross-pollina- 
tion most commonly occurs." 

According to Dunning (8), after the introduction and establish- 
ment of bumblebees in New Zealand red clover seeded abundantly, 
but previous to this time he says it seeded very little. The Agri- 
cultural Gazette of New South Wales (9, 16) maintains that bumble- 
bees were introduced into New South Wales from New Zealand so 
that they would be able to produce clover seed for home use, which 
up to this time was largely imported. At Failford, New South 
Wales, red clover seeded abundantly (35), although no bumblebees 
had been noticed in that vicinity. The pollinating was thought to 
have been done by several native insects. This was several years 
after the introduction of bumblebees. Later (17) it was stated that 
bumblebees had become well established. 

Waldron (41, 42) found in his experiments that bumblebees were 
responsible for about 95 per cent of red-clover seed and that a small 
quantity may be produced by natural self-pollination. 

Mliller (29, p. 184-186) states that when a bee draws its proboscis 
out of a clover flower cross-pollination is assured and self-pollination 
may also take place, but that the self-pollination is probably neutral- 
ized and superseded by the immediately preceding cross-pollination. 

Folsom (10, p. 116) considers the Italian race of honeybees as 
important as the bumblebees in clover-seed production, while Arm- 
strong (1) claims that honeybees are able to extract nectar from 
red-clover flowers in New Zealand. Pammel (31, p. 172) shows that 
honeybees are able to collect pollen from red-clover flowers and 
thereby cross-pollinate them. Robertson (33, p. 177) states as 
follows: "But while butterflies may sometimes effect cross-fertili- 
zation of the red clover, they are of doubtful value, if not injurious. 
* * * But butterflies can insert their thin tongues without 
depressing the keel, and, even if they get a little pollen on their 
thin proboscides, it is apt to be wiped off by the closely approxi- 
mated tips of the petals, which close the mouths of the flowers." 



EED-CLOVER SEED PRODUCTION. 5 

OUTLINE OF POLLINATING EXPERIMENTS. 

It is a well-known fact that the yield of clover seed varies greatly 
from year to year, and no distinct correlation with any marked 
climatic factors has been determined. It was thought that 
possibly the absence of suitable pollinating insects, such as bumble- 
bees, might in some seasons be responsible for the reduced yields 
of seeds. This is especially true when conditions were such that 
there was no other apparent reason for the failure of the crop to 
set seed. In order to obtain light on this point, a series of experi- 
ments was outlined to determine (1) whether clover flowers were 
able to set seed without the assistance of outside agencies; (2) 
whether clover flowers were able to set seed when their own pollen 
was transferred to their stigmas by outside agencies; and (3) the 
relative efficiency of the honeybee and the bumblebee as cross- 
pollinators of red clover. 

In order to overcome any local environmental factors, the experi- 
ments were conducted at Ames, Iowa, and La Fayette, Ind., and were 
repeated to some extent at the Arlington Experiment Farm, Va. 
The work on individual clover heads was performed on heads pro- 
tected from the action of insects by tarlatan cloth. This cloth has 
about twice as many meshes to the linear inch as ordinary mosquito 
netting. Wliere numerous plants were to be protected from all 
outside agencies, cages of wire screen having 14 meshes to the 
linear inch were used. In some instances, where it was desired to 
permit the entrance of all insects smaller than bumblebees, cages 
made of galvanized-wire screen having four meshes to the linear mch 
were employed. 

All work was done on second-crop red clover unless otherwise spe- 
cifically stated. 

STRUCTURE OF THE RED-CLOVER FLOWER. 

The heads of red clover contain from 35 to 150 flowers each, and 
according to Pammel and Clark (30) the average number per head 
for black loam soil at Ames, Iowa, is 71.1 for the first crop and 9S.1 
for the second crop 

The flowers of red clover consist of a greon pubescent calyx with 
five pointed lobes and an irregular magenta or purple corolla of live 
petals (Fig 1 .) The claws of the petals are more or less united to 
the staminal tube This stammal tube is formed by the union of the 
filaments of the nine inferior stamens To the greater portion of the 
anterior end of this common tube, formed by the uniting of the claws 
of the petals with the stamina! tube, is attached the broad base of 
the vexiUum. The carina, which is composed of two petals united 
at one edge, is attached to the uiferior part of the edge of the tube 



6 BULLETIN 289, U. S. DEPAETMENT OF AGRICULTURE. 

hot ofciipied by the vexillum. Even though the base of the carhia 
is narrow it is able to return to its normal position shortly after being 
bent downward. The alae are attached by their flexible claws to the 
common tube. Before a flower opens the alse are pressed closely to 
the carina, although as the flower matures they spread apart. The 
staminal tube splits superiorly to admit the tenth free stamen. The 
filament of this superior stamen lies along the side of the staminal 
tube and therefore does not interfere with the proboscis of a bee 
which is inserted to collect nectar. Nectar is secreted at the bases 
of the stamens and accumulates in the staminal tube around the base 




Fig. 1. — Different parts of a red-clover flower: 1, Anterior view of flower; 2, posterior view of flower after 
tlie vexillum has been removed; 3, posterior view of flower, showing the carina, which has been forced 
apart (twice the magniflcation of the other drawings); 4, right ala, from within; 5, ri;;ht half of carina, 
from without, the claws of 4 and 5 haying been partly broken off; 6, the essential organs emerging from 
the depressed carina; 7, longitudinal section of a flower, o, Calyx; b, tube formed by the partial union 
of 9 filaments with the claws of the vexillum, alae, and carina; c, vexillum; d, concave part of the umer 
side of ala; e, lower border of ala, bent outward; /, outward surface of ala; g, pouched swellmg on the 
baseofanala; ft, carina; i, style; fc, superior free stamen; Z,stigma; 7n, anthers; m, point of union between 
alpe and carina; o, point of flexure of the carina; p, part of the upper border of ala, bent outward; q 
downward extension of the vexillum; r, staminal tube; s, style; t, ovary. (After Miiller in part.) 

of the ovary. The filaments which compose the staminal tube sepa- 
rate in the hollow of the carina. Each filament bears a fertile anther. 
The pistil is inclosed within the staminal tube, the upper part of the 
style and stigma of which are inclosed with the anthers in the carina. 
The stigma is situated slightly above the stamens in most flowers, 
although in some the anther of the longest stamen is as high as the 
stigma. 

When a bee inserts its proboscis into the staminal tube, it is inserted 
between the vexillum and the carina. In doins: this the carina and 



RED-CLOVER SEED PRODUCTION. 7 

aliB are pressed downward and the stigma and anthers arc thrust up 
against the bee's head. Since the carina and stamens are elastic, the 
pollen is thrown with considerable force against the head of the bee. 
When the bee releases the pressure on the carina and alae, the parts 
return to their normal position on account of the elasticity of the 
base of the carina and a small dilated vesicular process at the base 
of each ala. (Fig. 1.) 

LENGTH OF THE COROLLA TUBE OF RED-CLOVER FLOWERS. 

The corolla tube of red clover is stated by Knuth (22, v. 2, p. 289) 
and Miiller (29, pp. 184-186) to be from 9 to 10 millimeters in length. 
Pammel and King (32) report an average length of 9.4 mUlimeters 
for 450 flowers. Schachinger, according to Fruwirth (12, pp. 163- 
166), says the corolla tubes are shorter in the second crop than in 
the first crop, and for this reason smaller bees are able to work on the 
second crop than on the first. 

Fifteen corolla tubes from each of 28 heads of first-crop red clover 
were measured at Ames, Iowa. The greatest variation found in 
different flowers of the same head was 2 millimeters. The 420 
corolla tubes varied from 8.5 millimeters to 11.5 millimeters, with 
an average length of 9.6 millimeters. 

DEVELOPMENT OF THE FLOWERS OF RED CLOVER. 

The stamens of rod clover develop much more rapidly than the pistU, 
and the length of the longer set exceeds that of the pistil until near 
the time the flower opens. The pollen is formed in the longer stamens 
tlirough the division of the mother cells when the pistil is about 0.25 
millimeter in length. The division in the pollen mother cells of the 
shorter stamens closely follows that in the longer stamens. Wlicn 
the pistil is about 1 millimeter in length, only about one-tweKth of 
its length at maturity (fig. 2, A), the pollen grains are apparently 
mature so far as their size, their shape, and the thickness of their 
walls are concerned. At this stage the two ovules are well formed, 
but the egg and endosperm cells are not developed till later and are 
not ready for fertilization until just previous to the opening of the 
corolla. The later development of the poUen consists in protoplasmic 
changes. After the pollen grains have reached their mature size and 
their walls have become mature the protoplasm shows very little or 
no granular nature. Just before the flowers open the protoplasm 
becomes very dense. At this stage the protoplasm contains much 
oil in the form of an emulsion. The poflen will now germinate. 

The pistil has a stylar canal reaching from the ovary almost to 
the stigma. Just previous to and during the opening of the corolla 
the pistil elongates more rapidly than the stamens, and as a result 
the stigma is usually pushed beyond the anthers in the open flower 



8 



BULLETIN 289, U. S. DEPAETMENT OF AGRICULTURE. 



(fig. 2, B). The stigmatio surface is papillate and has a fringed 
appearance in the mature flower. The papillae contain much oil and 
have rather heavy walls, which react to the test for cut in. 

Both ovides develop embryo sacs (fig. 2, C). Fertilization usually 
takes place in each ovule; but only one, so far as observed, matures 
into a seed. Should plants occur that mature both ovules, there 
would be an opportunity to produce strains with twice the seed- 
yielding capacity of those now grown. 




Fig. 2. — Red-clover flowers, showing different stages of development. A. — Lengthwise section of a red- 
clover flower at an early stage (X50): a, Calyx tube; 6, stamina! tube; c, standard; d, one of the long 
stamens; e, anthers of two long stamens; /, free stamen; g, stigma; h, the two ovules; i, anther of a short 
stamen; j, stylar canal. B. — Lengthwise section of an open, flower, showing the character of the stigma 
and its position relative to the anthers (X2S): a, Stigma; b, anthers of two long stamens; c, anthers of 
two short stamens. C— Lengthwise section through the base of a flower, open and ready for fertilization 
(X40): a, Egg; 6, endosperm cell; c, calyx; d, staminal tube; e, nectar glands;/, free stamen. D. — A 
median, longituduial section through the nucellus of a sterile ovule which should have been ready for 
fertilization, the flower being open; all cells remained vegetative and no reproductive cells were produced 
(XlS). E. — Pollen grain (X325): g, Germ pore; n, nucleus; w, wall. 

INFERTILE OVULES OP RED CLOVER. 

Infertile ovules are a common occurrence in red clover and occur to 
a considerable extent tlu-oughout the season. A section through the 
nucellus of an infertile ovule is shown in figure 2, D. In the infertile 
ovules all colls remain vegetative and no embryo sac is formed. The 
largest percentage of infertility has been found to occur in first-crop 
red clover, and this infertility appears to accompany moist soil and 
atmospheric conditions. During the first crop many plants produce 



EED-CLOVER SEED PRODUCTION, y 

100 per cent of infertile ovules. With siicli pltints the presence of 
bees is not a matter of importance, for the ovules have no reproduc- 
tive cells; hence there can be no fertilization and no production of 
seed. During the second crop, when the season is generally dry and 
favorable for seed setting, there is some infertility, ranging from a 
low percentage or none in some plants to a high percentage in others. 
It is very probable that this infertility of ovules is to a greater or less 
degree a hereditary character and that the production of a high- 
yielding strain will consist, among other features, in selecting those 
plants with the least tendency toward infertility. 

POLLEN OP RED CLOVER. 

The pollen grains of red clover are almost globular wIkui turgid, 
with a little flattening at the germ pores. Wlien measured in a 
25 per cent cane-sugar solution the pollen grains have an aA^erage 
size of 44.5 by 43 ix (fig. 2, E). Tlie grains are not fully turgid when 
shed from the anthers and one diameter in each is shortened and 
the other diameter lengthened by an infolding of the wall. In this 
condition Martin (25) found the average dimensions of 100 pollen 
grains to be 26 by 48 ix, while Miss Clark (30) found the average size 
of 1,024 pollen grains to be 31.7 by 56.29 /;,. 

Wlien dropped in water the pollen grains take it up very rapidly 
and burst almost instantly. On account of this feature of the poUon 
there can be little effective pollination when the flowers are wet. 
Pollination at night or in the morning when the flowers are wet 
with dew is not likely to be effective. 

Germination of the pollen of red clover was found by Martin (25) 
to depend upon a proper water supply. Good artificial germination 
can be secured on parchment paper or animal membranes which are 
just moist enough to permit the pollen to absorb the requisite amount 
of water for germination. Germination takes place within a limited 
range of variation in the water supply, and it is only by trials of 
wetting and drying that the proper moistm*e content of the mem- 
branes may be secured. Under proper conditions of moisture and 
temperature, germination takes place usually in 8 to 10 minutes. 

FUNCTION OF THE STIGMAS OF RED-CLOVER FLOWERS. 

Microchemical tests of the stigmas of red-clover flowers show 
no sugars or starches present. An oily emulsion, however, does 
occur in the papillge. Crushed stigmas placed on animal membranes 
had no apparent effect on the germination of the pollen or on the 
directions of the tubes. 

When pollen is deposited on the stigmas it lodges between the 
papilla, takes up water, and soon becomes turgid, but the water 
supply is so regulated by the stigmas that no bursting occm's. It 
2990°— Bull. 289—15 2 



10 BULLETIN 28a_, U. S. DEPARTMENT OF AGEICULTUEE. 

is probable that the only function of the stigmas in the germination 
of the pollen is thaf of supplying the requisite amount of moisture 
to the poUen. If such is the function of the stigmas, a wet soil or 
humid atmosphere, both of which tend to increase the water content 
of the stigmas, may allow the pollen to absorb too much water and 
thus prevent fertilization. Martin (25) found pollen lying dormant 
on stigmas 18 hours after pollination during cool, moist weather. 
This dormancy might have been due to the effect of low temperature 
upon the poUen alone, but could have been due to an interference 
with the moisture adjustmejit. 

FERTILIZATION OF RED-CLOVER FLOWERS. 

TIME REQUIRED BETWEEN POLLINATION AND FERTILIZATION. 

The time between the pollination and the fertilization of red-clover 
flowers varies. Flowers pollinated in July, when the temperature 
was high and killed 18 hours after pollination, showed that in most 
cases fertilization had taken place. In October, when the tempera- 
ture was much lower, the time between poUination and fertdization 
ranged from 35 to 50 hours. 

NECESSITY OF FERTILIZATION OF RED-CLOVER FLOWERS. 

It has been reported that red clover is able to develop seed without 
fertilization; but field experiments, as well as laboratory tests, have 
disproved this statement. One of the most noticeable features of 
this work was the fact that aU the flowers of heads which were 
covered with tarlatan before they came into bloom and left in this 
condition untd they withered remained in full bloom from 9 to 10 
days. Flowers of red clover wither shortly after fertilization takes 
place. This is why red clover heads usually contain flowers in bud, 
in bloom, and withered at the same time. 

In order to further test the necessity of fertilization, a large number 
of heads were covered with tarlatan before any flowers came into 
bloom. An examination of more than 500 flowers at various times 
after they began to wilt showed no embryos. The ovules were 
disintegrating. 

POTENCY OF POLLEN IN SELF-POLLINATION. 

In order to determine the potency of poUen in self-pollinated 
flowers of red clover, a number of heads were covered with tarlatan 
two or three days previous to the opening of the flowers. Some of 
these covered flowers were seK-poUinated by springing the carinas, 
while the rest were cross-poUinated by springing the carinas and 
applying poUen from flowers on other plants to their stigmas. By 
mounting the pistils of these flowers in a 30 per cent sucrose solution 



RED-CLOVER SEED PRODUCTION. 11 

and flattening thorn witli a little pressure on the cover glass, the 
pollen tubes could be traced through the stylar canals, as pollen tubes 
have a denser and more granular content than the cells of the style. 

An examination of 30 flowers which had been self-pollinated for 55 
hours showed good germination on the stigmas but no fertilization. 
The number of pollen grains germinating on the stigmas ranged 
from 3 to 25 in each of the 30 flowers. The tubes had made a slow 
growth and none exceeded 4 millimeters in length. An examination 
of 20 flowers which had been self-pollinated for 90 hours showed that 
one pollen tube had attained a length of 7.5 millimeters, while the 
others were 5 millimeters or less in length. At this rate of growth 
the longest tube would have required about 48 hours more to reach 
[he ovules, or about six days to traverse the entire distance from 
stigma to ovule. Flowers examined four days after springing the 
carinas showed the eggs in a disintegrated condition. It is therefore 
probable that in case of self-pollination the pollen tubes do not reach 
the ovules in time to effect fertilization. 

An examination of the 30 flowers which had been cross-pollinated 
for 55 hours showed that fertilization had taken place in all of them. 

CROSS-POLLINATION AND SELF-POLLINATION OF RED CLOVER. 

The results published by previous investigators on the cross- 
pollination and the self-pollination of red clover do not agree. These 
investigators appear to be about ec^ually divided as to whether red- 
clover flowers are self-fertile or not. The experiments of Frandsen, 
according to Lindhard (23), Fruwirth (12, p. 163-166), and others 
show that red-clover heads which were covered during their blooming 
period and not pollinated failed to set seed Frandsen and Fruwirth 
also show that pollen must come from an entirely separate plant 
in order to fertilize the ovules of red-clover flowers. On the other 
hand, Garton, according to McAlpine (24) states that self-poUination 
is as common with red clover as it is with the bean. 

The relative efficiency of the bumblebee and honeybee as cross- 
pollinators of red clover has also been discussed by scientific investi- 
gators, as well as by agricultural papers and bee keepers. Bee men 
generally agree that the Italian race of honeybees can extract nectar 
from red-clover flowers. Little has been said, however, about the 
ability of the honeybee to coUect poUen from red clover. 

In view of the above diverse opinions in regard to the self-pollination 
and the cross-pollination of red clover, a number of experiments were 
outlined in order to determine (1) whether red-clover flowers were 
self -fertile; (2) if self-fertfle, whether any effective method of self- 
pollination could be found which would be applicable for use on a field 
scale; and (3) the relative efficiency of the bumblebee and honeybee 
as cross-poUinators of red clover. 



12 BULLETIN 289, U. S. DEPARTMENT OP AGRICULTURE. 

ARTIFICIAL MANIPULATION OF CLOVER HEADS. 

Experiments were eondueted to determine, if possible, the effect 
on seed production of various types of artificial manipulation of the 
clover heads while the flowers were in bloom (fig. 3) A sufficient 
numl>er of heads were selected on each plant so that the work could 
be conducted on heads covered with tarlatan (fig. 4) and on heads 
exposed to the action of insects. The experiments on the heads 
exposed to the action of insects were to determine whether the 
artificial manipulation of the flowers would have any harmful effect 
on seed production. The different treatments given the heads 
covered with tarlatan were to determine whether fertilization could be 




Fig. 3.— a screen cage (iu the background) in wliich bumblebees were confined. Hand-pollinalion work 
is in progress in the foreground. 

produced by any method of artificially manipulating clover flowers 
from which insects were excluded. For this work, plants were 
selected bearing at least eight heads which would come into bloom 
at approximately the same time. These plants were taken at random 
and each marked with a stake, as shown in figure 5. The heads on 
each plant were labeled from A to H, inclusive, and treated as shown 
in Table I. 

These experiments were conducted in Iowa, in 1911 on 50 plants 
at Ames, and 25 at Altoona, and in 1912 on 70 plants at Ames. 

Table II gives the results obtained on 25 representative plants, 
selected from the entire number, and also the average seed yield per 
head of the entire 145 plants experimented with in 1911 and 1912. 



EED-CLOVER SEED PRODUCTION. 13 

Table I. — Treatment of clover blossoms in the artificial vianipulation experiments. 



Head. 


Heads not covered with laiialan. 


Heads covered with 
tarlatan. 


A... 


Check 




B... 


Entire head rolled between thumb and finger . . 




C 

D 


Keel of each flower sprung with a toothpick, care being taken to rub the 
pollen on each stigma. (A separate toothpick was used for each head.) 
Tapped several times with coarse toothbrush 




E... 




Check 


r.... 






o... 






H. 













Tablk II. — Effect of different types of artificial manipulation upon, the seed produclion 
of red-clover plants treated in 1911 and 1912. 



2.5 Selected liEPRE.sENXATivE Plants. 





Number of seeds produced jier head. 


liocation, year, and desi,i;nati()n of 
plants. 


Heads not covered with tarlatan. 


Heads coverec 


with tarlatan. 




A 


R 


C 


D 


E 


F 


G 


n 


Ames, 1911: 

No. 1 


.33 

40 
4(3 
40 

.54 
90 
44 
47 
82 


26 
46 

69 
47 
IS 
33 
57 
26 
55 


30 
39 
31 
20 
13 

'43 
62 
16 
59 


47 
26 
35 
36 
68 
56 
69 
40 
28 
65 







2 
1 













1 














3 




No. 2 




No. 3 




No. 4 




No. 5 




No. 6 




No. 7 




No. 8 




No. 9 . . 





No. 10 











52.8 


37.7 


36.5 


47.0 


0.3 


.1 


.3 


9 






Altoona, 1911: 
No. 11 


26 
10 
29 
9 
22 


13 
16 
12 
6 
16 


24 
12 
4 
11 

28 


5 
38 

7 
14 











1 










1 








No. 12 


1 


No. 1:3 




No. It 




No 15 








Average 


19.2 


12.6 


15.8 


12.8 





.2 


.2 









Ames, 1912: 

No. 16 


20 

33 

39 

20 

44 

52 

37. 

40 

42 

15 


3 
31 
45 

40 , 
40 
61 
28 ■ 
35 
28 

5 ' 


25 

24 

" 24 

28 

24 

. 51 

33 

40 

26 

6 


15 
36 
43 
21 
28 
52 
35 
21 
43 
12 
















2 



■ 













1 










No. 17 





No. 18 - 





No. 19 





No. 20 . . . . 





No. 21 





No. 22 





No. 2.3 





No. 24 





No. 25... ...;. 


1 








34.2 


31.6 


28.1 


30. 6 





■- 


. 1 


. 1 







Summary of Average Resitlts for the Entire 145 Plants. 



Ames, 1911: 50 plants 


44.3 
16.0 
48.9 


38.8 
9.4 
42.5 


3.5.1 
20.3 
41.3 


42.3 
18.0 
41.5 


0.08 
.28 
.1 


0.16 
.48 
.1 


0.16 
.24 
.14 


0.22 


Altoona 1911: 25 plants 


.36 


Ames, 1912: 70 plants 


.15 






Average, 145 plants 


41.6 


35.5 


35.5 


37.7 


.12 


.18 


.16 


.21 







The average seed yields given in the first part of Table II should 
be compared with the average seed production shown in the sum- 



14 



BULLETIN 289, U. S. DEPARTMENT OF AGRICULTURE. 



maiy of the same table. The 25 phxnts hsted separately were selected 
to represent all the plants upon which this experiment had been 
conducted in 1911 and 1912. Wliile the average results shown by 
the selected plants vary somewhat from the results of all the plants, 
still they are representative of the plants as a whole. It will bo noted 
that the seed production of the uncovered heads varies considerably 
on the same plant, so that fmal results must be taken from the average 
of treated heads on a number of plants rather than on a few plants. 
For this reason the results given in the summary more nearly represent 
true conditions than those given in the first part of Table II. 

From the results obtained on the heads not covered with tarlatan 
it will be seen that artificial manipulation was detrimental to seed 
production, since the average yield of the check is higher than that 




Fig, 4.— Heads of red clover covered with tarlatan to prevent pollination by insects. 

of any treated series. This is undoubtedly due to the fact that the 
flowers were somewhat mutilated during the operation. Very little 
seed was obtained from the heads which were kept under cover and 
artificially manipulated. The few seeds obtained were probably the 
result of cross-pollination by bumblebees when the tarlatan cloth had 
been pushed against the heads by rain or had been cut by grass- 
hoppers. Rains would wash the starch from the tarlatan, thus per- 
mitting it to fall against the clover heads and allowing the flowers to 
protrude. This was avoided by either straightening out the cloth 
after it had dried or re-covering the heads. A few flowers on some 
heads, however, were exposed to the action of insects for a very 
short time. In the work which was conducted at Altoona, Iowa 
the grasshoppers were so bad that some heads had to be re-covered 



EED-CLOVER SEED PRODUCTIOK. 



15 



as often as tliree times a day. Many uncovered heads were partly 
destroyed by the grasshoppers, and this undoubtedly accounts for 
the small seed yield of the uncovered heads, since bumblebees were 
plentiful. 



HEADS COVERED AND NOT POLLINATED. 



Another experiment was conducted m order to determine whether 
clover heads kept covered during their entire blooming period and 
not pollinated could set seed. 

Plants having at least six heads which would come into bloom at 
approximately the same time were selected for this work. Fifty 




Fig. 5.— General view of the field in which the clover work was conducted in 1912 at Ames, Iowa. The 
stakes represent plants selected for individual pollination work. The cages in the background were 
used to test the efficiency of different insects as pollinators of red clover. 

plants at Ames and 25 at Altoona were selected in 1911 and 27 
plants at Ames in 1912. The average seed yields per head are shown 
in Table III. 

Table III. — Average seed yields of clover heads which were covered with tarlatan and not 

pollinated. 



Location, year, and number o 


plants. 




Heads 


covered with tarlalan. 




A 


B 


C 


D 


E 


F 


Ames, 1911: 50 plants 


0.1 

.16 



0.11 
.4 
.1 


0.15 
.35 





.04 



0.16 
.26 

.02 





Altoona, 1911: 25 plants 


.2 


Ames, 1912: 27 plants 











Average, 102 plants 


.08 


.17 


.15 


.009 


.14 


.04 







To the results presented in Table III may be added the results 
given in column E of the summary of Table II, where 145 heads were 



16 BULLETIN 280, U. S. DEPARTMENT OF AGRICULTUKE, 

covorcd, not troatod, and used as cliecks in those experiments. 
From the 757 heads covered and not treated in 1911 and 1912 an 
average of 0.1 seed per head was obtained. The rehitively high 
average obtained at Altoona in 1911 may undoubtedly be accounted 
for by grasshoppers mutilating the tarlatan which was used to cover 
the heads. On this account heads were occasionally exposed to the 
action of insects for a short time. 

Since no more seed was produced by these 'heads than may be 
accounted for by insects working on the flowers when they were 
occasionally exposed for a short time on account of rains or grass- 
hoppers, we may say that clover flowers must be pollinated l)y some 
agency before any seed is produced. 

EFFECT OF SELF-POLLINATION. 

Another experiment was conducted in which the clover lieads 
were covered with tarlatan before any flowers opened and were kept 
covered, except while being worked, until mature. As soon as the 
flowers came into bloom they were self-pollinated by springing the 
keels of the flowers with toothpicks, care being taken to rub pollen 
upon each stigma. A separate toothpick was used for each head. 
In 1911, 125 heads were self -pollinated and 170 heads in 1912. An 
average of 0.16 seed per head was obtained in 1911 and an average 
of 0.09 seed per head in 1912. 

The results of this experiment show, as have previous experiments, 
that red-clover flowers must be cross-pollinated in order to set seed 
on a commercial basis. The amount of seed obtained is so small 
that it was probably the result of bees working through the tarlatan, 
although the cytological work reported upon in this bulletin shows 
that it is possible to have an occasional seed jjroduced from self- 
pollination. 

SEED PRODUCTION OF HEADS UNDER ORDINARY FIELD CONDITIONS. 

As a field check on the preceding experiments a numl)cr of heads 
were tagged in 1911 and 1912 and neither covered nor artificially 
pollinated. These heads were labeled in different parts of the field 
and Table IV shows the number of heads in each group and the 
average seed yield per head. 

Table IV. — Average seed yield of clover heads not covered or nrtificiaUy pollinated. 



Location and year. 



Ames, 1911... 
Do 

Do 

Altoona, Ifin. 
Ames, 1912. . . 



Number of 

heads 
collected. 



300 
532 
470 
150 
05 



Average 

number of 

seeds per 

head. 



50.1 
55.4 
50.9 
43.6 
53.4 



RED-CLOVER SEED PRODUCTION. 17 

The results given in Table IV are somewhat higher thaii the average 
seed yield of the uncovered check of the experiments summarized in 
Table II. It may be that the close proximity of the checks given in 
Table II to heads covered with tarlatan kept bees from making as 
many visits to those heads as they would otherwise have made. 

FLOWERS POLLINATED WITH POLLEN FROM ANOTHER HEAD ON THE SAME PRIMARY 

BRANCH. 

Since the amount of seed obtained in 1911 from seK-pollinated 
heads under cover was so small that it could be accounted for by bees 
working through the tarlatan, it was not deemed necessary to 
emasculate the flowers for cross-pollination work in 1912. With 
this in view a series of heads was covered before any of the flowers 
came into bloom and later pollinated with pollen from another head 
on the same primary branch, 20 flowers on each of 11 heads being 
pollinated in this manner. Not a single seed was produced. 

FLOWERS POLLINATED WITH POLLEN FROM A HEAD ON A DIFFERENT PRIMARY 
BRANCH OF THE SAME PLANT. 

Another experiment similar to the preceding one was conducted, 
except that the pollen was taken from heads on different primary 
branches of the same plant, 20 flowers on each of 10 heads bemg 
pollinated in this manner. One seed was produced. 

CROSS-POLLINATION EXPERIMENTS. 

Alternately with the above two experiments 20 flowers on each of 
13 heads were pollinated with pollen from a separate plant. An 
average of 14.3 seeds per head was obtained. 

The results obtained in the last three experiments, as well as with 
all preceding ones, show that clover is practically self -sterile and that 
pollen must come from a separate plant in order to effect fertilization. 

BUMBLEBEES AS CROSS-POLLINATORS OF RED CLOVER. 

In view of the consensus of opinion that the bumblebee is responsi- 
ble for the cross-poUination of red-clover flowei-s, and since no 
investigator, so far recalled, has denied its abUity to do this, it was 
deemed desirable to study the relative efficiency of the bumblebee 
as a cross-poUinator of this plant. 

For this work a cage 12 feet sc|uare and 6 feet high, made of wne 
screen having 14 meshes to the linear inch, was erected shortly after 
the first crop of clover had been cut. As soon as the second crop 
started to bloom bumblebees were caught with an msect net and 
placed in the cage. It was soon found that bees would live about 
tlu-ee days when confined in the cage and that six bees in confinement 
would visit approximately as many flowers as one bee would have 
visited had it worked nearly all the time. With this in mind, two 
bumblebees were placed in the cage each forenoon until all the clover 



18 BULLETIN 289^ U. S. DEPARTMENT OF AGRICULTURE. 

heads were mature. An area 4 feet square was marked off iii this 
casre as soon as the clover was mature. From this area all heads 
were collected, kept separate, and thrashed by hand. Of the 311 
heads collected from this area an average of 30.4 seeds per head 
was obtained. 

Repeated field observations in Iowa in 1911 and 1912 showed that 
bumblebees were actively engaged in collecting nectar from eight to 
nine hours a day. Little work was done by them before the dew had 
entirely disappeared from the foliage and flowers or after 6 o'clock 
in the evening. Observations showed that bumblebees are able to 
pollinate 30 to 35 flowers a minute. However, they seldom visit 
more than eight to ten on a single head at one time. 

These results agree closely with those of Pammel and King (32), 
who state that bumblebees poUinate on an average 30 flowers a 
minute, and Smith, according to Beal (3), who estimates from counts 
that old bees wiU visit 35 flowers a minute and young bees seldom 
more than eight. 

HONEYBEES AS CROSS-POLLINATORS OF RED CLOVER. 

The ability of the honeybee to cross-pollinate red clover has been 
discussed by scientific investigators and beekeepers for some time. 
Those who do not believe that the honeybee is able to poUinate red 
clover base their statements for the most part on the fact that the 
proboscis of the honeybee is not long enough to reach the nectar 
located at the base of the staminal tube. Some investigators and 
bee men state that some strains of the Italian race of honeybees are 
able to obtain some nectar from red-clover flowers, while other 
investigators say that honeybees collect poUen from red-clover 
flowers and thereby cross-pollinate them. 

According to Knuth (22, v. 2, p. 289) the proboscis of the honeybee 
is 6 mm. in length, which is 3.6 mm. shorter than the average length 
of the corolla tubes of first-crop red-clover flowers. Honeybees may 
be able at times to obtain some nectar from the sides of the staminal 
tubes of red-clover flowers when a large amount is secreted or when 
the flowers are not in an upright position. Knuth (22, v. 2, p. 289) 
observes that Bombus terrestris, a species of bumblebee found in 
Europe, pierces the tubes of clover flowers and that honeybees later 
obtaiu nectar through these slits. Bomhus terrestris has a proboscis 
from 7 to 9 mm. in length. While working on the experiments 
reported upon in this bulletin several corolla tubes were observed 
which had been slit at the base, but it can not be stated that these 
slits were made by bees. Schneck (34) states that the Virginia car- 
penter bee {Xylocopa virginica) slits the lower end of the coroUa 
tubes of red-clover flowers and that he has observed honeybees 
obtaining nectar through the slits. 



EED-CLOVER SEED PRODUCTION. 



19 



111 order to determine the efficiency of the honeybee as a cross- 
pollinator of red clover, a cage 12 feet square and 6 feet high, made 
of galvanized-wire screen having 4 meshes to the linear inch, was 
erected in the same field as the bumblebee cage. It was previously 
determined that a mesh of this size would permit a honeybee, or any 
msect smaller than a honeybee, to pass through, but would not per- 
mit bumblebees to do so. Two weeks before the clover came into 
bloom a small colony of honeybees was placed in one corner of this 
cage (fig. 6). The bees soon learned to pass through the screen. By 
the time the clover began to bloom the bees had become accustomed 
to the cage, and while most of them worked on flowers outside, some 
could always be seen at work on the clover within the cage. Bees 




Fig. 6.-A screen cage in which a hive of honeybees was placed, in order to determine the efficiency of these 
insects as pollinators of red clover. 

working on the clover within the cage were observed to collect pollen 
from the flowers and carry it to the hive. 

As soon as all the flowers in the cage were mature, an area 4 feet 
square was measured off and all heads within this area were collected, 
kept separate, and thrashed by hand. Of the 623 heads collected 
from this area an average of 37.2 seeds per head was obtained. 

The higher yield of seed obtained in the honeybee cage than in the 
bumblebee cage may be attributed, at least in part, to the larger num- 
ber of bees which had access to this clover. However, the ratio of 
honeybees to bumblebees was no greater in the cages than in the 
clover fields in the vicinity of Ames in 1911. 

In 1911 the precipitation at Ames was 2.48, 3.83, and 0.39 inches 
below normal for June, July, and August, respectively. When the 



20 BULLETIN 289, U. S. DEPARTMENT OF AGRICULTUEE. 

clover was in l)loom very few nectar-producing plants were to be 
found. Wliether the honeybee would work on red clover to this 
extent in a year of normal rainfall when the number of other nectar- 
producing plants is larger is problematical, but our observations 
and results show that the honeybee is able to spring the keels of red- 
clover flowers and thereby cross-pollinate them. 

MECHANICAL CROSS-POLLINATORS OF RED CLOVER. 

A machine so constructed that a platform of brushes could be made 
to strike clover heads with a vertical stroke was placed on the market 
under the name of a clover cross-pollinizing machine (fig. 7). This 
machine received some favorable comment. In view of this fact, 
a number of experiments were outlined to test its efficiency and also 




Fig. 7. — Clover cross-polliiilzi ig machine. 

to test the efficiency of different types of hand-operated brushes as 
mechanical cross-pollinators of red clover. Some plats of red clover 
were treated with various types of brushes at different times of day, 
while other plats were treated when the clover heads were in different 
stages of bloom. The direction of the strokes with the brushes was 
also varied in order to see whether this would have any effect on 
the yield of seed. 

MACHINE POLLINATION EXPERIMENTS. 

In order to determine the efficiency of a clover cross-poUenizing 
machine several experiments were performed at Ames in 1911. 
Machines similar to the one used were offered for sale on the market 
at the time these experiments were being conducted. 



BED-CLOVER SEED PRODUCTION. 



21 



This niaehine was so constructed as to give vertical strokes with a 
brush 4 feet wide and 6 f(>et long. The brushes could be removed 
with little trouble and replaced with others of a different type. Two 
types of brushes were used in these experiments. Brush No. 1 was 
composed of palmetto fiber, the bristles of which were rather stiff 
and of a dark reddish-brown color. Brush No. 2 was composed of 
rice-root fiber of light color, somewhat more flexible than the pal- 
metto fiber. The plats treated were 12 feet wide and 100 feet long. 

The number of heads collected from each plat represents the num- 
ber contained within two hooped areas. These hooped areas were 
obtamed by sailing a hoop into the air so that it would light on a 
particular plat. Since each plat contained a uniform stand it was 
thought that by this method heads would be selected which would 
be representative of the entire plat. Table V shows the results 
obtained m these experiments. 

Table V. — Average seed yield of red clover on plats which vjere given various treatments 
with a cross-pollinizing machine. 





Treatments. 


Heads 
collected. 




No. 


Brush 
used. 


Times 
gone over. 


Thne 
between. 


Time 
of day. 


Number 
of days 
given. 


Seeds 
per head. 


Plat 1 


No. 1.... 
No.l.... 
No.l...- 


1 

2 


Days. 
1 
1 
3 


A.M.... 
A.M.... 
A.M.... 


7 
4 


4(50 
424 
499 
490 
414 
458 
5S2 
527 
487 
655 
492 


63.3 
59.4 
64.2 
65.1 
59.2 
70.3 
6(). 7 
70.9 
64.7 
64.0 
67.4 


Jlat 2 


Plat 3 


Plat 4, check 


Plat 5 

Plat 6 

Plat 7, check 


No. 1.... 
No.l.... 


2 


3 
3 


A.M.... 
A.M.... 


4 

4 


Plat 8 


No. 2.... 
No.l.... 


3 
3 


3 
3 


A. M.... 
P.M.... 


4 
4 


Plat 9 


Plat 10, check 


Plat 11 


No. 2.... 


3 


3 


P.M.... 


4 





It will be seen from the above experiments that the treatments 
with brush No. 1 decreased the yield 1.9 seeds per head, while the 
plats treated with brush No. 2 gave an increased yield of 3.9 seeds 
per head over the average of the check plats. 

EFFICIENCY OF DIFFERENT HAND-OPERATED BRUSHES AS MECHANICAL CROSS - 
POLLINATORS OF RED CLOVER. 

In order to test the efficiency" of hand-operated brushes as a means 
of mechanical pollmation of red clover, eight pairs havmg different 
types of bristles were used. The different pairs were labeled A, B, 
C, etc., for convenience in reference. Following is a brief descrip- 
tion of the different pairs of brushes used: 

(A) Rice-root fiber, somewhat stiff; bristles about 2 inches long and nearly erect. 

(B) Rice-root fiber, similar to A, but with bristles 3 inches long, somewhat coarser 
and more spreading. 

(C) Tam])ico fiber, finer but stiffer than the rice-rout fiber in brushes A and li, 

(D) Wire-bristle hairbrushes. 



22 BULLETIN 289, U. S. DEPARTMENT OF AGRICULTURE. 

(E) Indian palmetto fiber, coarser tlian any of the others, but somewhat brittle. 
A portion of this brush was modified by cutting out sections of the bristles to make 
a more uneven surface. 

(F) Ordinary bristle hairbrushes. 

(G) Same as brush E, but with about half of the bristles clipped out. 
(H) Same as brush C, but with about half of the bristles clipped out. 

The idea of utilizing pairs of brushes with different types of fibers 
was to determine, if possible, if any of them would give sufficient 
promise to warrant an application of the particular type of brush 
to a mechanically operated machme that would imitate the action 
of the small brush when operated by hand. For this reason no 
brushes were used which could not be duplicated on a machine for 
operation on a field scale. 

The work with the hand-operated brushes was done principally at 
La Fayette, Ind., and Ames, Iowa. In some experiments the heads 
were manipulated with the brushes at different times of day, while m 
other experiments different numbers of treatments were given the 
heads at varying intervals. The direction from which the heads were 
struck also varied in certain experiments, some being given vertical 
strokes and others lateral strokes. It was thought that cross-pollina- 
tion might be brought about by the vertical stroke, which apparently 
would enable some of the brush bristles to spring the keels and convey 
pollen from one flower to another. It was also thought that if the 
flowers were self-fertile the lateral strokes would accomplish this. 
The representative tables that follow indicate the prmcipal features 
brought out by this series of experiments. 

RELATIVE EFFICIENCY OF BRUSHES WHEN THE CLOVER HEADS WERE STRUCK 

HORIZONTALLY. 

In a clover field 2^ miles east of La Fayette, Ind., 26 square-rod 
plats were laid off for this experiment in 1911 (Table VI). AU of 
the heads in bloom at the time the plats were marked off were re- 
moved. 

Plat 1 was left as a check, no brushes being used on it. Plat 2 was 
worked with brushes A, one brush being taken in each hand and the 
blossoms struck between the brushes by a quick movement of the wrists. 
When in full bloom the heads received one treatment, the operator 
going only in one direction across the plat. Brushes B, C, D, E, and 
F were used on plats 3, 4, 5, 6, and 7, respectively, in the same manner 
as for brushes A on plat 2. Plat 8 was treated by going one way 
across it at right angles to the first way, thus giving each blossom two 
treatments, the strokes of the two treatments thus being at right 
angles to each other. Brushes A were used. Plats 9, 10, 11, 12, and 
13 each received treatment similar to plat 8, but with brushes B, C, 
D, E, and F, respectively. Plats 14 and 15 were left as checks. 
Plats 16, 17, 18, 19, 20, and 21 each received two treatments with 



EED-CLOVER SEED PEODUCTION. 



23 



brushes B, C, E, B, C, and E, respectively, in the same manner as 
plat 8. 

Two days later plats 16, 17, 18, 19, 20, and 21 each received two 
more treatments with brushes B, C, E, B, C, and E, respectively, and 
three days later plats 19, 20, and 21 each received an additional two 
treatments with brushes B, C, and E. 

Plats 22 to 26, inclusive, were square-rod plats selected at intervals 
in the field immediately surrounding the portion laid off with the 
regular plats. These were designed to give a, large number of check 
plats to show the variation in the field under ordinary conditions. 

At harvest time 500 heads were picked at random from each of the 
26 plats. These heads w^ere huUed and the average seed production 
for tlie 500 heads is given in Table VI. 

Table VI. — Average seed yield per head obtained when clover heads were struck horizontally 
■with different types of brushes and at different intervals at La Fayette, Ind., in 1911. 



No. ■ 


Strokes per 
treatment. 


Brush 
used. 


Date of 
treatment. 


Heads 
collected. 


Seeds 
per head. 


Plat 1, check 








500 
500 
500 
500 
500 
500 
500 
500 
500 
500 
500 
500 
500 
500 
500 
500 
500 
500 
500 
500 
500 
500 
500 
500 
500 
500 
4,000 


SS 2 


Plat 2 


2 
2 
2 
2 
2 
2 


A 
B 
C 
D 
E 
F 
A 
B 

D 
E 
F 


August 2--. 

do 

do 

do -- 

do 

do 

do 

do 

do 

do 

do 

do 


46 


Plat 3 


67 1 


Plat 4 


50 1 


Plat 5 


27 


Plate 


3Q 4 


Plat? 


34 7 


Plats 


44 


Plats 


35 4 


Plat 10 


59 4 


Plat 11 


27 2 


Plat 12 


34 7 


Plat 13 


59 9 


Plat 14, check 


49 3 


Plat 15, check 








57 


Plat 16 


2 
2 
2 
2 
2 
2 


B 
C 
E 
B 

C 

E 


August 2, 4-- 

do 

do 

August 2, 4, 7 

do 

do 


59.9 


Plat 17 


26 9 


Plat 18 


64.4 


Plat 19 


35.4 


Plat 20 


32.0 


Plat 21 


22.5 


Plat 22, check 


28 6 


Plat 23, check 








59.0 


Plat 24, check 








49 5 


Plat 25, check 








43.2 


Plat 26, check 








31.6 










47.0 













Table VI shows that only 6 plats averaged higher than the average 
of the checks, whereas 12 plats averaged lower than the average of 
the checks. A variation ahuost as wide is shown in the 8 check 
plats as in the 18 treated plats, yet the results show that, as a whole, 
the brushes injured the seed production. Tlie low results on the 
plats treated with brush D, the wire hair brush, were undoubtedly 
due to the fact that the wire bristles penetrated and injured the 
flowers. 

This brush work was duplicated on a small scale with brushes A, 
B, and E on a few heads left uncovered and on heads that were 
covered with tarlatan, near La Fayette, in the summer of 1913. 
Tlie heads were worked once. Tlie results are given m Table VII. 



24 



BULLETIN 2S!>, U. S. DEPARTMENT OF AGRICULTURE. 



Table VII. — Average seed yield of clover heads which were struck horizontally tvith different 
types of brushes and either protected or unprotected from, insects at La Fayette, Ind. , in 
1913. 



Brush used. 


Strokes 
per 
treat- 
ment. 


Heads left inicov- 
ered. 


Heads covered with 
tarlatan. 


Heads 
worked. 


Seeds per 
head. 


Heads 
worked. 


Seeds per 
head. 


A 




3 
5 
5 
3 
5 
5 


37.4 
3.S. S 
50.1 
GO.S 
57.5 
GO. 1 


1 
1 

2 
1 
1 
2 





B 





E 





Check... 





Do 





Do 










Table VII shows that in each case of the heads left uncovered the 
check produced the most seed. As in Table VI, the brushes appar- 
ently reduced the seed production. It will be noted also that both 
the brush-treated and untreated heads produced no seed when 
protected from insect visitation. 

Experiments to test the relative efficiency of horizontal strokes 
with the different brushes were also conducted at Ames, in the sum- 
mer of 1911. Ten plats 4 feet square were used. These plats were 
marked off by placuig stakes at the corners and comiecting these 
stakes with heavy cord. Plats 1 to 8 were each given one treatment 
on each of three consecutive days, with brushes B, A, E, C, F, D, G, 
and H, respectively, and plats 9 and 10 were left as checks. Duph- 
cate tests, using the same brushes, respectively, on plats 37 to 44 
were also made. The results are shown in Table VIII. 

Table VIII.^^4yem,(7e seed yield per head obtained when clover heads were struck horizon- 
tally with different types of brushes at Ames, Iowa, in 1911. 





Strokes 
per treat- 
ment. 


Treat- 
ments. 


Orif;mal tests. 


Duplicate tests. 


Brush used. 


Plat 

No. 


Heads 
worked. 


Seeds 
per head. 


Plat 

No. 


Heads 
worked. 


Seeds 
per head. 


B 




3 
3 
3 
3 
3 
3 
3 
3 


1 

3 
4 
5 
tj 

s 
9 
10 


454 
47« 
457 
322 
440 
435 
320 
430 
532 
470 


44. 8 
41.1 
42.6 
46.2 
43.9 
36.2 
45.1 
41.0 
55.4 
50.9 


37 
38 
39 
40 
41 
42 
43 
44 
9 
10 


438 
515 
490 
485 
470 
445 
460 
432 
532 
470 


44.9 


A 


43.2 


E 


30.1 


c .. ■ 


42.5 


F 


47.5 


D 


33.0 


G 


39.9 


H 


42.9 


Check 


55.4 


Do 






50.9 











Table VIII shows that in every case where brushes were used the 
seed production fell below the yield of the check plats. 

From the results obtained in Tables VI, VII, and VIII it is con- 
cluded that at least horizontal strokes with the brushes in question 
reduced the seed production on account of the flowers being muti- 
lated by the brushes, 



BED-CLOVER SEED PKODUCTION, 



25 



RELATIVE EFFICIENCY Ol'^ 



BRUSHES WHEN THE 
VERTICAI,LY. 



CLOVER HEADS WERE STRUCK 



In order to test the efficiency of ])rushes in promoting cross-pollina- 
tion by carrying j^ollen frt>ni one plant to another on the bristles of 
the brushes, experiments were conducted at Ames, Iowa, in the 
summer of 1911 on plats 13 to 20 with different pairs of brushes. A 
vertical stroke on each of three mornings, tlu"ee days apart, was 
given. The plats were 4 feet square. At maturity all heads from 
each plat were collected, kept separate, and later tlirashed. 

The experiments on "plats 21 to 28 were the same in all respects, 
except that one treatment when the flowers were in early bloom, 
instead of three treatments, was given each plat. Plats 9 and 10 
wore used as checks. The results are presented in Table IX. 

Table IX. — Average seed yield per head obtained when clover heads were struck vertically 
with different types of brushes at Ames, Iowa, in 1911. 



Bnish usod. 


Strokes 
per 
treat- 
ment. 


flats 


given three treat- 
ments. 


Plats given a single treat- 
ment. 


riat 

No. 


Heails 
worked. 


Seeds per 
head. 


Plat 
No. 


Heads 
worked. 


Seeds per 
head. 


Check 




9 
10 
13 
14 
15 
16 
17 
18 
19 
20 


.532" 
170 
3tiG 
490 
440 
.521 
416 
476 
.510 
462 


.5.5. 4 
.50.9 
37.0 
42.0 
42.1 
45.3 
39.3 
37.7 
41.8 
44.7 


9 
10 
21 
22 
23 
24 
25 
26 
27 
28 


532 
470 
442 
440 
420 
415 
380 
400 
430 
436 


.55.4 


Do 




50 9 


B 




46.8 


A... 


33 8 


E 


45.0 


C. 


38 9 


¥.... 


54 5 


D 


45.9 


G 


48.1 


H 


49.0 







Table IX shows that with the exception of plat 25 the treatment 
considerably reduced the yield below that of the lowest yielding check 
plat. The results of the treated plats 13 to 20, inclusive, taken as a 
whole show a decrease of four seeds per head less than the yield of 
plats 21 to 28, inclusive. This may be accounted for by the fact that 
plats 13 to 20, inclusive, received two more treatments with the 
brushes than plats 21 to 28, inclusive, and were therefore subject to 
more injury from the bristles of the brushes. It will be seen from 
these experiments that the vertical strokes with the brushes proved no 
more efficient than the horizontal strokes in the production of seed. 

RELATIVE EFFICIENCY OP BRUSHES WHEN PRESSED TOGETHEIi BELOW THE CLOVER 
HEADS AND PULLED UPWARD. 

Experiments were conducted in the summer of 191 1, at Ames, Iowa, 
to determine the efficiency of pressing a pair of brushes together below 
the clover heads and pulling them upward with considerable force, 
but stiU not enough to break off the heads. The plats were 4 by 4 
feet in size. Pair A of the brushes was used. Thi"ee treatments 



26 



BULLETIISr 289, U. S. DEPARTMENT OF AGRICULTUKE, 



thi'ee days apart in the forenoon were given. The results are pre- 
sented in Table X. 

Table X. — Average seed yield per head u'hen brushes were pressed together heloir the clover 
heads and pulled uprvard, summer of 1911. 



No. 


Heads 
worked. 


Brush 
used. 


Treat- 
ments. 


Seeds 
per head. 


Plat 9, check 


532 
470 
432 
460 






55.4 


Plat 10, check 






50.9 


Plat 11 


A 
A 


3 
3 


39.2 


Plat 12.. 


40.9 







Table X shows that this manner of treatment as well as the hori- 
zontal and vertical stroke treatments caused a decrease in the pro- 
duction of seed. ' 

SUMMARY. 

A study of the cytology of red-clover flowers shows that many of 
them contain infertile ovules. The percentage of infertile ovules is 
greater in the first crop than in the second crop. In the first crop 
many plants produce 100 per cent of infertile ovules, while in the 
second crop the percentage of infertility ranges from none to a high 
figure. The percentage of infertile ovules in red clover is probably 
correlated with moisture conditions. 

The pollen grains of red clover are very sensitive to moisture. 
On account of this, there can be little effective pollination when the 
flowers are wet. Germination of the pollen grains takes place only 
within a limited range of variation in the water supply. It is prob- 
ably true that the only function of the stigma is that of supplying the 
requisite amount of water to the pollen for germination. 

The time between pollination and fertilization varies with the 
temperature of the atmosphere. The time between pollination and 
fertilization in July is approximately 18 hours, while in October it 
varies from 35 to 50 hours. An examination of 30 flowers which had 
been self-poUinated for 55 hours showed good germination on the 
stigmas but no fertilization. The pollen tubes made a slow growth 
and none exceeded 4 mm. in length. In flowers which had been 
self-poUinated for 90 hours one pollen tube attained a length of 
7.5 mm., while the rest were 5 mm. or less in length. The pistils of 
red clover average about 12 mm. in length. Eggs were found to be 
disintegrating four days after the flowers opened. 

The self -pollination and cross-pollination experiments which were 
conducted in the field checked up very closely with the results 
obtained from the cytological studies. The average yield of seed 
obtained on heads which were not pollinated and on heads which 
were self-pollinated in different ways was less than one-half of 1 per 



EED-CLOVER SEED PRODUCTION. 



27 



cent. This small yield of seed may be accounted for by the occa- 
sional access of bees to these heads for a very short time, on account 
of rains or grasshoppers mutilating the tarlatan which was used to 
cover the heads. 

The bumblebee is an efficient cross-pollinator of red clover. 
Bumblebees are able to pollinate from 30 to 35 flowers a minute. 

The honeybee proved to be as efficient a cross-pollinator of red 
clover as the bumblebee in 1911. Wlien the precipitation was con- 
siderably below normal in June, July, and August, 1911, and but 
few nectar-producing plants were to be found, honeybees collected 
large quantities of pollen from red clover. In order to collect pollen 
they must spring the keels of the flowers. In doing this they cross- 
pollinate the flowers. 

A clover cross-poUinizing machine which was offered for sale on 
the market did not prove to be an efficient cross-poUinator of red 
clover. 

The various types of hand-operated brushes which were used did 
not prove efficient as cross-pollinators of red clover. In nearly all 
cases where these brushes were used the seed yield was decreased 
instead of increased. This was undoubtedly due to the bristles of 
the brushes injuring the flowers, since the average seed yield of the 
plats which received three treatments with the brushes was lower 
than that of the plats which received but one treatment. 



LITERATURE CITED. 

(1) Armstrong, J. B. 

1883. The fertilization of the red clover. In Gard. Chron., n. s., v. 20, no. 516, 
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(2) Beal, W. J. 

1887. Gra.sses of North America, v. 1. New York. 

(3) 

1907. Planning an experiment to show to what extent bumblebees aid in pollin- 
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1907, p. 136-138. 

(4) BOLLEY, H. L. 

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(5) Cook, A.J. 

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(6) Darwin, C. R. 

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New York, 482 p. 

(7) 

1898. The Origin of Species ... v. 1. New York. 

(8) Dunning, J. W. 

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(9) Fertilizing clover and cow-grass. 

1891. In Agr. Gaz. N. S. AVales, v. 2, pt. 10, p. 636. 

(10) FOLSOM, J. W. 

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(11) Fruwirth, Karl. 

1906. Enclosing single plants and its effect on a large number of important agri- 
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(12) 

1906. Die Ziichtung der landwirtschaftlichen Kulturpflanzen. Bd. 3. Berlin. 

(13) Genevier, Gaston. 

1876. Inflorescence et fecondation dans le genre trifolium. In Assoc. Fraiig. 
Avan. Sci. Compt. rend., 4th sess., 1875, p. 726-730. 

(14) Hopkins, A. D. 

1896. On the flowering habits of timothy and red clover and the pollenization of 
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Sci., 1896, p. 35-40. 

(15) ■ ^- 

1896, Some notes on observations in West Virginia on farm, garden, and fruit 
insects. In U. S. Dept. Agr. Div. Ent. Bui. 6, n. s., p. 71-73. Also in 
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29 



30 BULLETIN 289^ U. S. DEPARTMENT OF AGEICULTUEE 

(16 



(17 
(18 
(19 
(20 
(21 

(22 
(23 

(24 
(25 
(26 
(27 

(28 

(29 
(30 

(31 

(32 
(33 
(34 

(35 



HUMBLEBEES. 

1892. In Agr. Gaz. N. S. Wales, v. 3, pt. 1, p. 78. 

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1897. In Agr. Gaz. N. S. Wales, v. 8, pt. 5, p. 353. 
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1909. Pollination of clover. In Wallace's Farmer, v. 34, no. 43, p. 1347. 
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1895. The Natural History of Plants ... [tr.] from the German ... v. 2. London. 
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Knuth, p. E. 0. W. 
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LiNDHARD, E. 

1911. Om R0dkl0verens Best0vning og de Humlebiarter, som herved er virk- 
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McAlpine, a. N. 

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Martin, J. N. 

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1914. Comparative morphology of some Leguminosse. In Bot. Gaz., v. 58, 
no. 2, p. 154-167, pi. 8-11. 

Martinet, G. 

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160-169, illus. 
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Asa Gray and others. In Proc. Acad. Nat. Sci. Phila., 1876, p. 108-112. 

MtJLLER, Hermann. 

1883. The Fertilization of Flowers. London, 669 p., illus. 

Pammel, Edna C, and Clark, Clarissa. 

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Pammel, L. H. 

1903. Ecology. Carroll, Iowa, 360 p., illus., pi. 

and King, Charlotte M. 

1911. Pollination of clover. Li Proc. Iowa Acad. Sci., 1911, p. 35-45, illus. 

Robertson, Charles. 

1892. Flowers and insects. In Bot. Gaz., v. 17, no. 6, p. 173-179. 

Schneck, Jacob. 

1891. Further notes on the miitilation of flowers by insects, hi Bot, Gaz., v, 

16, no. 11, p. 312-313. 
Seeding of red clover. 
1895. In Agr. Gaz. N. S. Wales, v. 6, pt. 6, p. 439. 



EED-CLOVER SEED PRODUCTION. 31 

(36) Shamel, a. D. 

1906. The effect of inbreeding in plants. In U. S. Dept. Agr. Year1)ook, 1905, 

p. 377-392, fig. 90-91, pi. 42-44. 

(37) SiRRINE. F. A. 

1891. Notes on methods of cros.s-pollination. In Iowa Agr. Exp. Sta. Bui. 13, 

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(38) Smith, C. B. 

1907. Red clover seed-growing. In Bailey, L. H. Cyclopedia of American Agri- 

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(39) Stebler, F. G., and Schroter, Carl. 

1889. The Best Forage Plants ... 3 v. in 1., illus. London. 

(40) . 

1913. Die Besten Futteipfianzen . . . Bd. 1. Bern. 

(41) Waldron, L. R. 

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(42) — 

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(43) Wallace, Henrv. 

1892. Clover Culture. Des Moines, Iowa, 156 p., illus. 

(44) 

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(46) WiTTE, Hernfrid. 

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